By Shirley McBride, Mary Heenan, Martin Clynes (auth.), Dr. Martin Clynes (eds.)
Cell tradition ideas permit various molecular and mobile organic inquiries to be addressed, supplying physiological stipulations when heading off using laboratory animals. as well as easy concepts, a variety of specialized useful protocols masking the subsequent parts are incorporated: telephone proliferation and dying, in-vitro versions for phone differentiation, in-vitro types for toxicology and pharmacology, business program of animal mobilephone tradition, genetic manipulation and research of human and animal cells in culture.
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Extra resources for Animal Cell Culture Techniques
Plaques of parainfluenza type 3 in VERO cells stained with 2 %(v/v) methylene blue (x20) 5. Remove the GM from the wells of the cluster plate. 6. 2 ml virus suspension (two wells for each dilution) and adsorb for 1 h at 37°C. Tilt the plate at IS-min intervals to prevent the monolayer from drying out and to ensure an even distribution of inoculum. 7. Wash the infected monolayers with prewarmed, sterile PBS to remove unadsorbed virus. 8. Remove the overlay medium from the water bath and allow to cool before pouring onto the monolayer.
25 g of sodium carbonate in 100 ml of distilled water. The stain may be stored for up to 12 months. An area of the agar adjacent to the suspect colony is swabbed with a cotton swab soaked in Dienes' stain. The stain will diffuse to the colony and the colony is then microscopically examined. Stained mycoplasma colonies have dense blue centers and light blue peripheries. Bacterial colonies are also stained but are decolorized in 30 min, in contrast to the mycoplasma colonies, which do not become decolorized.
3. 5 with 1 M HCI. 4. tm filter, and refrigerate until required. Note: Check pH perodically, as it is critical for optimal fluorescence of the DNA stain. 3 Screening for Mycoplasma Contamination in Animal Cell Cultures 43 Preparation of (overslips and Slides 1. Soak the required number of coverslips and slides in detergent for 1 h. 2. Rinse thoroughly in tap water for 5 min and in deionized water for a further 5 min. 3. Rinse the coverslips in methanol and wipe dry with lint-free tissue. The slides can be refrigerated in methanol until required (use a sparkproof refrigerator).
Animal Cell Culture Techniques by Shirley McBride, Mary Heenan, Martin Clynes (auth.), Dr. Martin Clynes (eds.)